There is increasing interest in developing an immunological approach to contraception for humans and sterilization for animal populations. A contraceptive vaccine would provide many advantages over currently available methods of contraception. Methods of contraception such as hormone therapies and chemical or mechanical barriers against fertilization have serious drawbacks, such as undesirable side effects and less than complete effectiveness. For example, side effects of hormonal therapies such as the pill include cancer, and in the case of mechanical barriers, increased susceptibility to infection. In addition, contraceptive vaccines would further be useful for fertility control of animal populations, where long-term or permanent sterilization, without the need for frequent intervention, is desirable. For example, such long-term sterilization would be useful for controlling fertility in human beings or agriculturally important livestock, such as cattle and pigs. Further, contraceptive vaccines would be useful for permanent sterilization regimes useful for pest control, such as for sterilization of rodents or other unwanted populations.
While there has been much interest in the development of immunocontraceptives, the focus has been, until recently, on the development of immunocontraceptives directed against sperm surface antigens, or on already known peptide hormones such as human chorionic gonadotropin and follicle stimulating hormone. One obstacle to the development of an effective egg surface antigen based immunocontraceptive vaccine has been the lack of knowledge regarding the molecular identities of egg surface proteins known to be directly involved in the fertilization process.
Mammalian fertilization may be defined as a series of gametic interactions in which capacitated sperm must first penetrate the cumulus cells and zona pellucida (the egg vestments), then bind to and fuse with the egg plasma membrane (oolemma). The initial binding event between gametes is known as primary binding and occurs, in the mouse model, when the zona pellucida protein, ZP3, binds to a receptor(s) on the sperm (reviewed in (McLeskey et al., 1998, Int. Rev. of Cytol. 177: 57-113). This binding event also initiates the acrosome reaction in which hydrolytic enzymes are released from the acrosomal compartment and act on the zona pellucida to facilitate penetration of the zona pellucida by sperm. Zona penetration is known as secondary binding and is mediated by the zona protein, ZP2, and one or more molecules on the inner acrosomal membrane (reviewed in Snell and White, 1996, Cell 85: 629-637).
Upon emergence from the zona pellucida, sperm then cross the perivitelline space and bind to and fuse with the oolemma. The molecular basis of sperm-oolemma binding and fusion has yet to be fully elucidated; however; recent evidence has demonstrated that integrins are involved in the interaction. Almeida et al. (1995, Cell 81:1095-1104) found that when oocytes were treated with monoclonal antibodies against the egg surface integrin α6β1, mouse sperm-oolemma binding was reduced. Further, these investigators reported that somatic cells which express α6β1 bind mouse sperm avidly while somatic cells that lack α6 or β1 do not. A proposed sperm surface ligand for α6β1 is fertilin. Fertilin contains a domain homologous to a family of integrin ligands known as disintegrins (Blobel et al., 1992, Nature 356: 248-252), which suggest a cell adhesion function for the molecule. Also, recombinant fertilin is known to bind to the oolemma (Evans et al., 1997, Dev. Biol. 187:79-93), with both monoclonal antibodies to fertilin (Primakoff et al., 1987, J. Cell. Biol. 104: 141-149) and fertilin peptide analogs (Almeida et al., 1995, Cell 81: 1095-1104; Evans et al., 1995, J. Cell. Sci. 108: 3267-3278) blocking sperm-oolemma binding and fusion.
Sperm-egg binding and fusion is likely to require multiple receptor-ligand interactions and other oolemmal proteins are likely to be involved in the fertilization process. In fact, there is indirect evidence implicating other oolemmal proteins in sperm-egg interaction. A purified sperm-associated protein (protein DE) which is involved in fusion in the rat, binds to the surface of zona-free rat oocytes (Cohen et al., 1996, Biol. Reprod. 55: 200-206). Another putative oolemmal sperm receptor is removed from the surface of radioiodinated mouse eggs following trypsin treatment and reappears on the egg surface after 3-6 h of culture (Kellom et al., 1992, Mol. Reprod. Dev. 33: 46-52). The reappearance of this 94 kDa protein on the egg surface coincides with the ability of the trypsin-treated eggs to be penetrated by sperm.
Glycosyl-phosphatidylinositol (GPI)-anchored proteins may play a key role in gamete interaction. GPI-anchored proteins possess a covalently linked glycosylated phosphatidylinositol moiety which serves to attach the protein portion of the molecule to the cell surface lipid bilayer (Low and Saltiel, 1988, Science, 239: 268-275). Proteins linked to the cell surface via a phosphatidylinositol anchor are known to be involved in a wide variety of cellular functions including T cell activation, hydrolysis of extracellular matrix proteins, transduction of extracellular stimuli, and cell-cell adhesion (reviewed in Low and Saltiel, 1988, Science, 239: 268-275). GPI-anchored proteins can be released from the cell surface by treatment of cells with the highly specific enzyme phosphatidylinositol-specific phospholipase C (PI-PLC) (Low and Finean, 1978, Bioch. Biophys. Acta. 508: 565-570). Therefore, treatment of intact cells with PI-PLC has become a useful tool to characterize the released proteins and to investigate the role of GPI-anchored proteins in cell function.
The hamster oocyte is unique in that zona-free eggs from other species such as the mouse, rat, and guinea pig do not incorporate heterologous sperm as readily (Yanagimachi, 1972, J. Reprod. Fertil., 28: 477-480; Hanada and Chang, 1976, J. Reprod. Fertil., 46: 239-241; and Quinn, 1979, 210: 497-506). Because of this promiscuity, the zona-free hamster egg has been used extensively in the sperm penetration assay (SPA) to assess the fertilizing capacity of human spermatozoa (Yanagimachi et al., 1976, Biol. Reprod., 15: 471-476; Rodgers et al., 1979; Liu and Baker, 1992, Fertil. Steril., 59: 698-699). In spite of the widespread use of this assay, the molecular interactions which occur between the human sperm and hamster oocyte during gamete interaction remain largely unknown. Presumably, however, there are molecules on the hamster egg plasma membrane (oolemma) which specifically interact with molecules on the human sperm plasma membrane during sperm-egg binding and fusion.
There have been a number of recent attempts to produce contraceptive vaccines directed against egg antigens (U.S. Pat. Nos. 5,820,863, 5,641,487, 5,637,300, and 4,996,297). To date, because of their relative abundance and accessibility to immunodetection, the focus has been on identifying zona pellucida epitopes. However, results from fertility trials in several species have shown that ovarian histopathology is often observed in ZP3 immunized animals. Therefore, commercial contraceptive companies have lost interest in zona proteins as contraceptive immunogens.
A new approach for an effective contraceptive vaccine that specifically targets antigens directly involved in the fertilization process is needed. However, to date, there are no reports of vaccines directed against egg protein(s) directly involved in the process of sperm-egg fusion step which is required for fertilization. The present invention is directed to contraceptive compositions and methods that are based on egg specific surface proteins.
Definitions
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
As used herein, “nucleic acid,” “DNA,” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
The term “peptide” encompasses a sequence of 3 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids. Peptide mimetics include peptides having one or more of the following modifications:                1. peptides wherein one or more of the peptidyl —C(O)NR— linkages (bonds) have been replaced by a non-peptidyl linkage such as a —CH2-carbamate linkage (—CH2OC(O)NR—), a phosphonate linkage, a —CH2-sulfonamide (—CH2—S(O)2NR—) linkage, a urea (—NHC(O)NH—) linkage, a —CH2-secondary amine linkage, or with an alkylated peptidyl linkage (—C(O)NR—) wherein R is C1-C4 alkyl;        2. peptides wherein the N-terminus is derivatized to a —NRR1 group, to a —NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)2R group, to a —NHC(O)NHR group where R and R1 are hydrogen or C1-C4 alkyl with the proviso that R and R1 are not both hydrogen;        3. peptides wherein the C terminus is derivatized to —C(O)R2 where R2 is selected from the group consisting of C1-C4 alkoxy, and —NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and C1-C4 alkyl.        
Naturally occurring amino acid residues in peptides are abbreviated as recommended by the IUPAC-IUB Biochemical Nomenclature Commission as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Norleucine is Nle; Valine is Vat or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any amino acid. Other naturally occurring amino acids include, by way of example, 4-hydroxyproline, 5-hydroxylysine, and the like.
Synthetic or non-naturally occurring amino acids refer to amino acids which do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein. The resulting “synthetic peptide” contain amino acids other than the 20 naturally occurring, genetically encoded amino acids at one, two, or more positions of the peptides. For instance, naphthylalanine can be substituted for trytophan to facilitate synthesis. Other synthetic amino acids that can be substituted into peptides include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl, beta.-amino acids, and isoquinolyl. D amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptides. Other derivatives include replacement of the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) with other side chains.
As used herein, the term “conservative amino acid substitution” are defined herein as exchanges within one of the following five groups:                I. Small Aliphatic, Nonpolar or Slightly Polar Residues:                    Ala, Ser, Thr, Pro, Gly;                        II. Polar, Negatively Charged Residues and Their Amides:                    Asp, Asn, Glu, Gln;                        III. Polar, Positively Charged Residues:                    His, Arg, Lys;                        IV. Large, Aliphatic, Nonpolar Residues:                    Met Leu, Ile, Val, Cys                        V. Large, Aromatic Residues:                    Phe, Tyr, Trp                        
As used herein, the term “purified” and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment.
As used herein, the term “MOP5 polypeptide” and like terms refers to polypeptides comprising SEQ ID NO: 2 and biologically active fragments thereof.
As used herein, the term “MOP8 polypeptide” and like terms refers to polypeptides comprising SEQ ID NO: 4 and biologically active fragments thereof.
As used herein, the term “biologically active fragments” or “bioactive fragment” of an egg surface polypeptide encompasses natural or synthetic portions of the native peptide that are capable of specific binding to at least one of the natural ligands of the respective native MOP5 or MOP8 polypeptide.
“Operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
As used herein, the tern “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.